Part:BBa_K774003
Comparator Circuit Part 2
Lack of specificity is a problem which many of the parts in the parts registry suffer from, and certainly a challenge which we faced when trying to detect nitrogenous species. From this potential problem spawned a potential solution; the Comparator Circuit. Part BBa K774003 is one of a pair of BioBricks which are designed to specifically bind to each other while ligated to two different promoters of overlapping specificity. This results in an integrating of the conflicting outputs of the two opposing gene systems.
Our system relies on two constructs that interact via complimentary base pair sequences both before and after the ribosome binding site of the reporter protein. The idea being that, when both transcripts are present in the chassis, they would bind together, inhibiting the translation of the reporter proteins.
Any imbalance of transcription due to the presence of the substrate of interest results in free mRNA of the gene system that detects that substrate. Crucially, if both promoters detect the same substrates but differ with one extra substrate being detected by one of the promoters, it is this substrate and this substrate only that our system will be able to detect in a simple and quantitative way.
Our team have constructed a countercurrent comparator circuit in which the reporter proteins are at the same end of the complimentary region, although a contracurrent system has been theorised. Both systems share a crucial subtractive nature comparable to an analogue computer. We envisage that, should the system be fine-tuned and expanded on, a variety of different business sectors from agriculture to spinoff pharmaceutical companies (such as the fictious Quanticare which you can read about on our wiki) could capitalise on this novel genetic technology.
What we have produced is a BioBrick pair that work in harmony, when ligated to promoters of interest and genes of interest, to sequester translation when both mRNA transcripts are present in the cell. The use of quantative tuners with these BioBricks is encouraged to ensure that the transcription rate both gene constructs are equal when both promoters are transcribing at their optimal rate. Although the parts have been submitted to the registry and theoretically characterised, time constraints have meant that further lab-based characterisation could not occur.
However, we hope to utilise any free time in our timetables during the next semester to characterise the BioBricks further (please see our project proposal), and hope that we will be given a chance to present our further findings at MIT!
To conclude, what we have created is a pair of antagonistic BioBricks that turned the pair of mRNAs in which they reside into translational repressor molecules when both are transcribed in tandem within a specific chassis of interest, a new application for mRNA complimentary base pairing within the registry and a project we feel could go very far indeed.
Theoretical Characterisation
The idea of the comparator circuit is to provide a modular method of signal integration that can produce a sensor which can specifically and quantitatively measure different chemical species within the cell using non specific promoters. Through mathematical modelling, an equation has been assembled which can predict the expression of each of the reporter proteins such as RFP and CFP.
Figure 2: Theoretical equation to predict the degree of expression of Construct 1 and 2. The full equation has been laid out in a way that is relevant only to Construct 1, however, the numbers can be reversed to be relevant to Construct 2. For ease of explanation, everything described will be relevant to Construct 1.
E = Proportion of expression rate of Construct 1 when both constructs are transcribed (i.e. there is knockdown of one construct) relative to the non-knocked down expression of Construct 1 when only Construct 1 is expressed.
A = The rate of transcription of Construct 1 as a proportion of the maximum transcription rate. As a proportion this is measured on a scale of 0 - 1. As an example if the rate of transcription is half of the maximum rate, rate would be 0.5 (arbituary units). It can be assumed the rate of transcription of construct 1 and 2 due to cellular components (e.g. RNA polymerase) is the same, however, the rate of transcription initiation will dictate the transcription rate. The initiation is reliant on the chemical species interacting with the transcription factor which binds to the promoter (i.e. nitric oxide,nitrates,nitrites to PyeaR). The '1' and '2' refer to the Construct 1 or 2 and hence the promoter and the measured fluorescent protein attached (e.g GFP, RFP, CFP, etc).
L = The length of the Construct 1 in the DNA form that is transcribed (i.e the leader and protein coding region).
Note: Leader refers to the section of RNA at the start of the mRNA that is not translated but has an effect on translation rate.
C = The rate of transcription. Assuming the rate of transcription of Construct 1 and 2 are the same because the same ribosomes and RBS are involved.
T = Half life of Construct 1 when only Construct 1 is present; the natural half life of Construct 1.
K = A constant of the biological system. This can only be measured through observation.
The full equation is modeled on the basic equation of:
where E is the rate of expression and E(A1) is the same as that explained above.
The additional complexity factors in less assumptions, and mimics a biological system, more closely. Below is a breakdown of the full equation.
This refers to the number of Construct 1 RNA transcripts undergoing transcription at any one time. The length of DNA is particularly important when the chassis is bacterial. In bacteria, as there is no true nucleus, translation occurs simultaneously with transcription. Transcription affects the probability of interaction between construct 1 and 2 and therefore, they are less likely to be translated. As the measurement of fluorescence is the output directly related to the rate of translation, the overall equation measures translation, however, translation rate is dependent on rate of transcription and degree of knockdown, and hence transcription is factored in here. L/C is the period of time taken for transcription to take place. It is the time in which translation can be initiated but it is unlikely that the two leaders will bind to one another
This part of the equation is the deduction of the knockdown of Construct 1 when there is Construct 2 expression and interaction. The biological constant, k, factors in that not all of construct 2 that is expressed will interact with construct 1 and vice versa. Hence, both exist despite construct 2 existing in small quantities. We believe that depending upon the assembly of the orientation of the two constructs within the plasmid, the interaction and hence the binding efficiency can be altered vastly. If the genes have opposite orientations, so that the termination sites are very close then the reduction of distance will increase the chances of interaction and hence make the sensory system more accurate.
This part of the equation encompasses the natural half life of Construct 1 when it alone is expressed (i.e. no expression of or interaction with Construct 2). As described before in the modelling from the basic equation, this is the lower part of the equation and puts it in perspective of Construct 1 and gives expression as a proportion of the maximum transcription. The half life is also Construct 1's half life.
So to bring it all together; the top half of the equation indicates the degree of translation of the RNA transcribed by the first promoter under any particular transcription rate of the two promoters in arbitrary units. To make this into a meaningful output it is divided by the maximum translation rate at that rate of transcription to equal E(A1); this indicates the degree of attenuation of one RNA from the other.
The Comparator Circuit has the potential to have real world applications, particularly in medicine (please see the future applications section on our wiki). To give an example, by monitoring blood sugar levels quantitatively via specific promoters, the sequence following the comparator circuit could encode for insulin. This could be transfected into human cells and could be used to alleviate the symptoms associated with type I diabetes. Moving back to the specific nitrogen sensor, attaching these promoters to the comparator circuit BioBricks and a gene for the synthesis of nitrogen reductase could result in a positive feedback loop to result in the tumour reducing in size. Macrophages naturally sense the presenBce of tumours in the body via their emission of nitric oxide. This could be taken one step further by adding nitrogen reductase to this system, where an excess of nitric oxide in the tumour environment. NO was seen to have cytotoxic properties in large amounts and, thus, a positive feedback loop could result in tumour apoptosis.
Sequencing
BLAST Analysis
To review the success of the sequencing of the Biorick sample, the predicted DNA sequence was compared to the sequence data using a Pairwise BLAST analysis ([http://blast.ncbi.nlm.nih.gov/Blast.cgi bl2seq]). The predicted DNA sequence was entered as the Query and the data from the sequencing analysis was entered as the Subject. The summary information at the top of the image identifies how many bases are identical between the two sequences. Lines joining the two sets of sequences indicate identical bases, and the absence of a line indicates a difference between the two sequences.
For this sample, the sequencing data is exactly as predicted.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 4
- 1000COMPATIBLE WITH RFC[1000]
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